Herpes simplex virus 2 (HSV-2) causes painful recurrent lesions in the genital and anal mucosa. Babies born to women shedding virus during delivery can become infected with HSV-2, often with devastating consequences. HSV-2 infects more than one out of six adults in the United States, and is a cofactor in acquisition of HIV. No vaccine to reduce or prevent HSV-mediated venereal infection and disease has yet been adopted. One potential vaccine reduced the HSV-2 infection rate in phase III trials but only in women, and only women who had not been previously exposed to HSV-1 (58% of adults are HSV-1 seropositive). The long-term objective of our research is to develop a universally effective prophylactic vaccine that limits infection and disease due to HSV-2. An effective vaccine against HSV-2 must be safe and highly immunogenic. Cells infected with ICP8- replication-defective virus express numerous viral proteins to stimulate immune responses, but progeny virions do not form and spread within the immunized host, making this a potentially safe and effective form of live virus vaccine. Previous and preliminary studies using ICP8-, replication-defective viruses indicate that 1) deletion of the gene encoding the virion host shutoff (vhs) protein, a viral RNase known to counteract host innate and adaptive immune responses, enhances the immunogenicity and efficacy of replication-defective HSV-1 and HSV-2 vaccines in mice;and 2) B7 costimulation molecules, which send a critical signal for activation of naive T cells, enhance the immunogenicity and efficacy of ICP8- HSV-2 when encoded from within the virus genome. We will address the hypothesis that replication-defective HSV-2 can be further modified to optimize its effectiveness by simultaneously mutating vhs and expressing B7 costimulation molecules. In Specific Aim 1, we will generate two new recombinant HSV-2 strains, ICP8-vhs- and ICP8-vhs-B7-2+. We will then compare ICP8- vhs-B7-2+ virus to ICP8-vhs- and ICP8-B7-2+ parental strains specifically to test whether the triple mutant optimizes induction of immune responses and capacity to prophylactically protect mice against HSV-2 genital infection. Another member of the costimulation molecule family, PD-L1, is upregulated during antiviral immune responses but inhibits function of memory and reactivating T cells by signaling through PD- 1. We further hypothesize that a booster immunization with a second vaccine that blocks negative regulatory signals mediated by PD-1/ligand interaction will augment immune responses to protect against genital disease caused by HSV-2. In Specific Aim 2, we will generate a recombinant HSV-2 strain that encodes soluble PD-L1, ICP8-vhs-sPD-L1+. Mice will be vaccinated with ICP8-vhs-B7-2+ virus and boosted with homologous ICP8-vhs-B7-2+ virus or heterologous ICP8-vhs-sPD-L1+ virus. We will determine whether blocking PD-1/PD-L1 interactions increases protective efficacy against HSV-2 genital infection. One or both of these new vaccine prototypes will then be ready for final evaluation prior to advancement to clinical trials. PUBLIC HEALTH RELEVANCE: Herpes simplex virus 2 (HSV-2) causes painful infections of the genital and anal mucosa and can infect babies during birth. HSV-2 infects one out of six adults in the United States, and has been identified as a cofactor for HIV acquisition. Our goal is to develop vaccine strains that can be used as prophylaxis to reduce disease burden due to primary and recurrent HSV-2 genital infections and to prevent transmission to newborns.